Transcript Forensic Serology, Biology, and DNA

Forensic Serology,
Biology, and DNA
Lecture 4
Forensic Serology

Serology = the study of antigen and antibody
reactions
 A, B, AB, O blood typing
 Rh+/ Karl Landsteiner (1901)
Whole Blood

Plasma (55%)
– Water (93%)
– Proteins (7%)
Fibrinogen
 Albumins, Globulins,
etc.
Electrolytes
Gases
Nutrients
Lipids
Waste
Hormones

–
–
–
–
–
–

Hematocrit (45%)
– Erythrocytes (99+%)
– Leukocytes
– Platelets
Serum

Plasma (55%)
– Water (93%)
– Proteins (7%)
Fibrinogen
 Albumins, Globulins,
etc.
Electrolytes
Gases
Nutrients
Lipids
Waste
Hormones

–
–
–
–
–
–

Hematocrit (45%)
– Erythrocytes (99+%)
– Leukocytes
– Platelets
Serum

Plasma (55%)
– Water (93%)
– Proteins (7%)
Fibrinogen
 Albumins, Globulins,
etc.
Electrolytes
Gases
Nutrients
Lipids
Waste
Hormones

–
–
–
–
–
–
Serum

Plasma (55%)
– Water (93%)
– Proteins (7%)
Fibrinogen
 Albumins, Globulins,
etc.
Electrolytes
Gases
Nutrients
Lipids
Waste
Hormones

–
–
–
–
–
–
Erythrocytes

Red blood cells
 Hemoglobin (Hb)
 Concave disk
– High surface to volume ratio

Facilitates diffusion of gases
 Proteins on surface (antigens)
Hemoglobin

Quarternary protein
 Heme groups (4)
 Iron (Fe) binds one
molecule of O2
Carbon Monoxide

Carbon monoxide (CO) also will bind (245x)
 Binds readily; leaves slowly
 Anemic hypoxia
CO Poisoning
Antigens

Millions on red blood cells
 A, B, and D antigens
Blood Type
Antigen(s)
Blood Type
Antigen(s)
A
Only A
Rh +
D antigen
B
Only B
Rh -
No D antigen
AB
Both A and B
O
Neither A nor B
Antibodies

Highly specific binding proteins
 Found in serum
 Anti-A, Anti-B, Anti-D
 Antiserum
 Bivalent binding sites
 Agglutination
Quick Summary
Blood Type
Antigen(s)
Antibody(s)
Frequency
A
Only A
Anti-B
42%
B
Only B
Anti-A
12%
AB
Both A and B
Neither Anti-A nor
Anti-B
3%
O
Neither A nor B
Both Anti-A and
Anti-B
43%
Remember:
Antigen
on RBC
Antibody
in serum
Forensic Serology

Stain (diluted in distilled water)
 Add anti-A serum (+/-)
 Add anti-B serum (+/-)
 Add anti-D serum (+/-)
Forensic Serology

Stain (diluted in distilled water)
 Add anti-A serum (+/-) = +
 Add anti-B serum (+/-) =  Add anti-D serum (+/-) = +
Result =
Antigen A and D = A, Rh+
Forensic Characterization of Blood
1)
2)
3)
Is it blood?
Is it human blood?
Can it be associated with blood from an
individual?
Is it Blood?

Hemoglobin:
– Presumptive tests
– Color tests (oxidation reactions)
 Benzidine (Adler) color test: blue = +
 Phenolphthalein (Kastle-Meyer) test: pink = +
 O-Tolidine (deriv. of benzidine): blue = +
 TMB (tetramethyl benzidine): blue = +
 LMG (leucomalachite green): green = +
Is it Blood?

Hemoglobin:
– Presumptive tests (Chemiluminescence/Fluorescence)
– Oxidation reactions (heme)
 Luminol: blue/green luminescence in the dark
–
–
–
–

Sodium Bicarbonate/carbonate mix
Fades within 30 seconds
Dilutes stain, follow up with phenolph., DNA?
VERY SENSITIVE (1:5 mil)
Fluorescein: 425-485 nm UV light (450 nm)
– Fluorescin to Fluorescein, with H2O2
– No DNA interference
Is it Blood?

Confirmatory tests
– Crystal tests
– DNA tests

BCA: phenolphthalein (& luminol)
Is it Human?

Precipitin test
– Rabbits injected with human blood
– Rabbits form anti-human antibodies (serum)
– Anti-human serum added to suspected human blood
 Ring Precipitin Test: test tube precipitate ring
 Ouchterlony Double Diffusion Test: gel plate “wells”
– Electrophoresis
– Other species (from rabbits)
Associate with an Individual

ABO typing
 Blood enzymes and proteins
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PGM (phosphoglucomutase) (1, 2-1, 2)
Hp, Hb, etc. each with varying frequency
Frequency (0.1 x 0.43 O type) = .043
DNA

DNA profile or DNA typing
 Deoxyribonucleic acid
 Cells contain chromosomes (nucleated)
 Chromosomes have genes
 Genes are DNA strands
 DNA double helix strands
 Nucleotides (4) are basic molecules
Base Pairing

Nucleotides (bases)
– Adenine (A)
– Guanine (G)
– Thymine (T)
– Cytosine (C)

A=T
G=C
 100 million b.p. on avg. chromosome
DNA Typing
1)
2)
3)
RFLP (Restriction Fragment Length
Polymorphisms)
PCR (Polymerase Chain Reaction)
STR (Short Tandem Repeats)
Restriction Fragment Length
Polymorphisms (RFLP)

Enzyme added to cut DNA in fragments
 Length of fragments will vary
 Separated (by weight) electrophoretically
 Treated with radioactive tags
 Visualized with X-ray photography
 Outdated, long, not as highly discrim., etc.
Polymerase Chain Reaction
(PCR)

“Grow” new DNA
– DNA replication
– Primer (known sequence) added
– Cycles (32 cycles, billions of strands)
 20-30 cycles (each about 2 minutes)
 1/50th of RFLP amount (nanogram)
– Known sequences of primers allow for typing
Short Tandem Repeats (STR)

Loci = locations on the chromosome
 Loci have repeating segments
 Very short (3-7 bases / 400 b.p. strand)
 Frequency of repeat strands:
–
–
–
–
–
Type (13 commonly used)
Length
Number of repeats
Numbers generated (2 = 1 from each parent)
Amelogenin gene (found on X and Y)
Mitochondrial DNA

DNA in nucleus or mitochondria
 Nuclear = chromosome = both parents
 Mitochondria = only maternal DNA
– BUT! More copies…higher sensitivity
– All maternal descendants have the genes

Hair, teeth, charred remains, nails, bones
 Application:



PCR
Sequencing (base pair order)
Slow, costly, not frequently done
DNA databases

CODIS: Combined DNA Index System
– 13 STR database
– Kits (Pro-Filer Plus, Co-Filer, etc.)
– Warrants (John Doe)
Other Considerations

Degradation of DNA
 Contamination of DNA



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Chemicals
Dilution
Mixed profiles
Controls
 Commercial kits (primers, loci, frequency)
 Population differences
 Statistical identification
Forensic Characterization
of Semen

Semen: seminal fluid + spermatazoa
– Fluid
 Nutrients, buffers, mucous, proteins, sugars
 Acid phosphatase (400x greater in semen)
– Sperm
 Tail (flagellum) and head (nucleus)
 Morphology
 Count
Forensic Characterization
of Semen

Presumptive test
– Acid phosphatase





Sodium alpha naphthylphosphate + Fast Blue B dye
Swab area, apply test
Blue = + for AP
Sensitivity 1/500 dilution
Confirmatory test
– PSA (prostate specific antigen) test

Apply anti-PSA serum to stain
– Presence of sperm cells
Other Biological Issues

Secretors (ABO antigens in other fluids)
 Saliva (amylase tests)
– 2 loci (AMY1, AMY2)
– Sweat also

Urine (urea and creatinine)
 Feces (bile byproducts, cells, blood)
Obtaining Samples

Sexual Assault Evidence Kits
– Swabs, blood, hair

Buccal swabs (knowns)
– Cheek cells

Bloody evidence
– DRY!!!
– Packaged separately
Hair Analysis

Traditionally: microscopy
– Occasionally: root with blood or cells
– Race, body origin

Now: DNA
– Typing from skin cell, blood (PCR, STR)
– From hair cells (mitochondrial)
Is it Human Hair?

Scales (cuticle)
 Cortex
 Medulla
Medulla

Types:
– Absent, fragmented, interrupted, continuous

Humans (head hair)
– Cylindrical
– Absent or interrupted very common
– Exception: Asians (continuous)

Medullary index
– Ratio of:
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


Diameter of medulla
Diameter of hair shaft
In humans, typically .33
In most other animals, .50 or greater